Group of alkaloids, the novel autophagic enhancers for treatment of cancers and neurodegenerative conditions thereof

ABSTRACT

The present invention discloses a method of treating cancer comprising administering an effective amount of an alkaloid, in which the alkaloid is liensinine, isoliensinine, dauricine, cepharanthine, hernandezine or thalidezine and isolated from the traditional Chinese medicinal herbs. The use of the alkaloid in treating neurodegenerative disorder is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. Ser. No. 14/562,781filed on 8 Dec. 2014, which claims benefit under 35 U.S.C. §119(e) ofU.S. Provisional Application having Ser. No. 61/923,231 filed 3 Jan.2014, which is hereby incorporated by reference herein in its entirety.

FIELD OF INVENTION

This invention relates to a group of novel autophagy enhancers, namelyliensinine, isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine, and their use thereof in treating cancers andneurodegenerative conditions.

BACKGROUND OF INVENTION

Autophagy is a cellular degradation process that involves the deliveryof cytoplasmic cargo such as long-lived protein, mis-folded protein ordamaged organelles, sequestered inside double-membrane vesicles(autophagosome) before entering lysosome for degradation. Autophagyoccurs at low basal levels in cells to maintain normal homeostaticfunctions by turnover of proteins and organelles. Upon cellularstressful conditions such as nutrient deprivation, oxidative stress,infection or protein aggregate accumulation, autophagy starts withmembrane isolation and expansion to form autophagosome that sequestersall unwanted cytoplasmic materials. Followed by fusion of theautophagosome with lysosome to form an autolysosome, all the engulfedmaterials are degraded to recycle intracellular nutrients and energy[1]. Both autophagy impairment and the age-related decline of autophagicfunction lead to the pathogenesis of many age-related diseases such asneurodegenerative disorders and cancers [2].

One of the key roles for autophagy is to degrade toxic aggregate-pronecytoplasmic proteins that are inaccessible to the proteasome when theyform oligomers or aggregates [3]; aggregate-prone proteins withpolyglutamine and polyalanine expansions, in turn, are degraded byautophagy [4]. Inhibition of mTOR induces autophagy and reduces toxicityof polyglutamine expansions or mutant proteins in fly and mouse models[4-7]. These mutant proteins include mutant α-synuclein which causesParkinson's disease, and polyglutamine-expanded mutant huntingtin thatcauses Huntington's disease [8,9]. In contrast, protein aggregates formin the cytoplasm when autophagy is inhibited in normal mice [10].Rapamycin, a United States Food and Drug Administration (FDA)-approvedimmunosuppressant, is found effective in treating fruit fly and mousemodels of Huntington's disease through increased autophagic clearance ofmutant huntingtin [5]. Besides, a small-molecule screen also revealednew chemicals that attenuate the toxicity of mutant huntingtin throughautophagy [9].

While autophagy may play a protective role in neurodegenerative diseases[9], autophagic dysfunction is associated with DNA damage, chromosomeinstability [11, 12], and increased incidence of malignancies [12].Modulators of autophagy may play a protective role through promotingautophagic cell death in tumors or augmenting the efficacy ofchemotherapeutic agents when used in combination. Several clinicallyapproved or experimental antitumor agents induced autophagy-related celldeath in various types of cancer cells [13-16].

Recently, natural compounds from alkaloids have been found to induceautophagy with potential neuroprotective or anti-cancer effects. Forinstance, alkaloids isolated from Chinese herbal medicine are importantsource for drug discovery [17]. Alkaloids such as berberine, matrine andtetrandrine, exhibit their anti-cancer effects through cell cyclearrest, apoptosis, autophagy, inhibition of metastasis or angiogenesis[18-20]. Camptothecin and vinblastine are chemotherapeutic drugs thathave been approved for clinical use [21,22]. In addition, alkaloids suchas isorhynchophylline [23] and berberine were also reported for theirneuroprotective effects in vitro.

SUMMARY OF INVENTION

In the light of the foregoing background, it is an object of the presentinvention to provide the alternate alkaloids as novel autophagyenhancers with their potential therapeutic application in cancers andneurodegenerative diseases by induction of autophagy-related cell deathin a panel of cancer cells and clearance of mutant huntingtin inneuronal cells. In one embodiment, such alkaloids include liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezine.

Accordingly, the present invention, in one aspect, is a method oftreating cancer including administering an effective amount of analkaloid to a subject in need thereof, wherein the alkaloid isisoquinoline alkaloid, bisbenzylisoquinoline alkaloid, biscoclaurinealkaloid or bisisoquinoline alkaloid.

In one exemplary embodiment, the isoquinoline alkaloid is liensinine;the bisbenzylisoquinoline alkaloid is isoliensinine, dauricine orhernandezine; the biscoclaurine alkaloid is cepharanthine; and thebisisoquinoline alkaloid is thalidezine.

In an exemplary embodiment, the cancer is cervical cancer, breastcancer, liver cancer, lung cancer or prostate cancer.

In another exemplary embodiment, the alkaloid exhibits specificcytotoxic effect towards a panel of human cancer cells.

In another exemplary embodiment, cancer is treatable byalkaloids-mediated autophagy; in a further exemplary embodiment, thealkaloids-mediated autophagy is autophagy-related gene 7 dependent.

In an exemplary embodiment, the cancer is caused by and/or originatedfrom cells containing wild-type autophagy-related gene 7, and is treatedby administering liensinine, isoliensinine, dauricine, cepharanthine,hernandezine and/or thalidezine to a subject in need thereof.

In yet another exemplary embodiment, the cancer is caused by and/ororiginated from apoptosis-resistant cells, and is treated byadministering isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine to a subject in need thereof.

According to another aspect of the present invention, a pharmaceuticalcomposition for treating cancer comprising an alkaloid is provided, inwhich the alkaloid is isoquinoline alkaloid, bisbenzylisoquinolinealkaloid, biscoclaurine alkaloid or bisisoquinoline alkaloid.

In one exemplary embodiment, the isoquinoline alkaloid is liensinine;the bisbenzylisoquinoline alkaloid is isoliensinine, dauricine orhernandezine; the biscoclaurine alkaloid is cepharanthine; and thebisisoquinoline alkaloid is thalidezine.

In an exemplary embodiment, the cancer is cervical cancer, breastcancer, liver cancer, lung cancer or prostate cancer.

In another exemplary embodiment, cancer is treatable byalkaloids-mediated autophagy; in a further exemplary embodiment, thealkaloids-mediated autophagy is autophagy-related gene 7 dependent.

In a further aspect of the present invention, a method of treatingneurodegenerative disorder including administering an effective amountof an alkaloid to a subject in need thereof is provided, in which thealkaloid is isoquinoline alkaloid, bisbenzylisoquinoline alkaloid,biscoclaurine alkaloid or bisisoquinoline alkaloid.

In one exemplary embodiment, the isoquinoline alkaloid is liensinine;the bisbenzylisoquinoline alkaloid is isoliensinine, dauricine orhernandezine; the biscoclaurine alkaloid is cepharanthine; and thebisisoquinoline alkaloid is thalidezine.

In an exemplary embodiment, the neurodegenerative disorder is caused bycells containing mutant huntingtin HDQ55/74.

In another exemplary embodiment, the neurodegenerative disorder isHuntington's disease.

In another aspect of the present invention, a pharmaceutical compositionfor treating neurodegenerative disorder comprising an alkaloid isprovided, in which the alkaloid is isoquinoline alkaloid,bisbenzylisoquinoline alkaloid, biscoclaurine alkaloid orbisisoquinoline alkaloid.

In one exemplary embodiment, the isoquinoline alkaloid is liensinine;the bisbenzylisoquinoline alkaloid is isoliensinine, dauricine orhernandezine; the biscoclaurine alkaloid is cepharanthine; and thebisisoquinoline alkaloid is thalidezine.

In an exemplary embodiment, the neurodegenerative disorder is caused bycells containing mutant huntingtin HDQ55/74.

In another exemplary embodiment, the neurodegenerative disorder isHuntington's disease.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1a to 1f show the chemical structures of liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezine.

FIG. 1g shows the results of cell cytotoxicity study of liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezinetowards a panel of cancer and normal cells.

FIGS. 2a to 2b show that liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine induce autophagic GFP-LC3puncta formation and autophagic protein LC3-II conversion in HeLa cancercells.

FIG. 2c shows that liensinine, isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine induce autophagic GFP-LC3 puncta formationin a panel of cancer cells.

FIG. 3 shows that liensinine, isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine-induced autophagy are abrogated byautophagic inhibitor, 3-methyl adenine (3-MA) in HeLa cancer cells.

FIGS. 4a to 4c show that liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine-induced autophagy aredependent on the presence of autophagy-related gene 7 (Atg7).

FIGS. 5a to 5b show that liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine activate autophagy throughmodulation of AMPK-mTOR signaling pathway.

FIGS. 6a to 6c show that liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine are able to induceautophagic cell death in wild-type Atg7 cells, but not in Atg7 deficientcells.

FIGS. 7a to 7e show that isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine are able to induce cell death inapoptosis-resistant cells.

FIGS. 8a to 8c show the cell cytotoxicity and clearance of HTT mutantHDQ55/74 of liensinine, isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein and in the claims, “comprising” means including thefollowing elements but not excluding others.

In this invention, a group of alkaloids including liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezineare identified as novel inducers of autophagy. The chemical structuresof these six alkaloids are demonstrated in FIGS. 1a to 1f respectively.Studies conducted by inventors demonstrate that liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezinecan induce autophagy and autophagic cell death in a panel of cancer andapoptosis-resistant cells. On the other hand, these compounds arecapable of promoting the degradation of mutant huntingtin with HDQ55 or74 CAG repeats in PC12 cells. Taken together, works by the inventorsprovide novel insights into the autophagic effect of selected alkaloidsand their potential uses in anti-tumor or neuroprotective therapy infuture.

Furthermore, in this invention, liensinine and isoliensinine are derivedand isolated from seed embryos of Nelumbo nucifera; dauricine is derivedand isolated from Asiatic Moonseed Rhizome; cepharanthine is derived andisolated from Stephania cepharantha; and hernandezine and thalidezineare derived and isolated from Thalictrum podocarpum Humb.

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

Example 1 Study on Cell Cytotoxicity

This example describes in vitro cell cytotoxicity of liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezine ina panel of human cancer and normal cells.

1.1 Cell Culture and Cytotoxicity Assay.

The test compounds of liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine were dissolved in DMSO at afinal concentration of 100 mmol/L and stored at −20° C. Cytotoxicity wasassessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay as described previously [25]. 4000-8000 HeLa (humancervical cancer), MCF-7 (human breast cancer), HepG2 (human livercancer), Hep3B (human liver cancer), H1299 (human lung cancer), A549(human lung cancer), PC3 (human prostate cancer) and LO2 (human normalliver) cells were seeded on 96-well plates per well. After overnightpre-incubation, the cells were exposed to different concentrations ofliensinine, isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine (0.039-100 μmol/L) for 3 days. Specifically, the followingconcentrations are used for all of the above alkaloids: 100, 50, 25,12.5, 6.25, 3.125, 1.5625, 0.78, 0.39, 0.195, 0.079, 0.039 ninon.Subsequently, 10 μL of MTT reagents was added to each well and incubatedat 37° C. for 4 hours followed by the addition of 100 μL solubilizationbuffer (10% SDS in 0.01 mol/L HCl) and overnight incubation. Absorbanceat 585 nm was determined from each well on the following day. Thepercentage of cell viability was calculated using the following formula:Cell viability (%)=Cells number treated/Cells number DMSO control×100.Data was obtained from three independent experiments.

1.2 Results:

As shown in FIG. 1g , significant cell cytotoxicity was observed withmean IC₅₀ value ranging from 4.52-61.8 μM observed in a panel of humancancer cells treated with liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine for 72 hours as revealed byMTT assay. However, the test compounds of liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine indicated no orinsignificant cytotoxic effect toward human normal liver LO2 cells.

Example 2 Study on Autophagic Effect

This example describes an in vitro study to demonstrate the autophagiceffect of liensinine, isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine.

2.1 Quantification of Autophagy GFP-LC3 Puncta.

GFP-LC3 puncta formation was quantified as previously described [15]. Inbrief, GFP-LC3 transfected cells grown on coverslips in a 6-well platewere treated with or without 20 μM of liensinine, 10 μM ofisoliensinine, dauricine, cepharanthine, hernandezine or thalidezine for4 hours, the cells were then fixed in 4% paraformaldehyde for 20 minutesat room temperature and then rinsed with PBS. Slides were mounted withFluorSave™ mounting media (Calbiochem, San Diego, Calif.) and examinedby fluorescence microscopy. The number of GFP-positive cells withGFP-LC3 puncta formation was examined under the Nikon ECLIPSE 80imicroscope. Representative images were captured with CCD digital cameraSpot RT3™ (Diagnostic Instruments, Inc., Melville, N.Y.). To quantifyfor autophagy, the percentage of cells with punctate GFP-LC3fluorescence was calculated by counting the number of the cells withpunctate GFP-LC3 fluorescence in GFP-positive cells. A minimum of 150cells from 3 randomly selected fields was scored.

2.2 Detection of Autophagic Marker Protein LC3 Conversion.

After treatments with liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine, cells were harvested andlysed in RIPA buffer (Cell Signaling Technologies Inc., Beverly, Mass.).The cell lysates were then resolved by SDS-PAGE. After electrophoresis,the proteins from SDS-PAGE were transferred to nitrocellulose membranewhich was then blocked with 5% non-fat dried milk for 60 minutes. Themembrane was then incubated with LC3 primary antibodies (1:1000) in TBSTovernight at 4° C. After that, the membrane was further incubated withHRP-conjugated secondary antibodies for 60 minutes. Finally, proteinbands were visualized by using the ECL Western Blotting DetectionReagents (Invitrogen, Paisley, Scotland, UK).

2.3 Quantification of Liensinine, Isoliensinine, Dauricine,Cepharanthine, Hernandezine and Thalidezine-Mediated Autophagy in thePresence of Autophagic Inhibitor.

GFP-LC3 puncta formation was quantified as previously described [15]. Inbrief, HeLa cells expressing GFP-LC3 were treated with 20 μM ofliensinine, or 10 μM of isoliensinine, dauricine, cepharanthine,hernandezine or thalidezine in the presence of autophagic inhibitor,3-methyl adenine (3-MA, 5 mM), for 4 hours. The cells were then fixed in4% paraformaldehyde for 20 minutes at room temperature and then rinsedwith PBS. Slides were mounted with FluorSave™ mounting media(Calbiochem) and examined by fluorescence microscopy. To quantify forautophagy, the percentage of cells with punctate GFP-LC3 fluorescencewas calculated by counting the number of the cells with punctate GFP-LC3fluorescence in GFP-positive cells. A minimum of 150 cells from 3randomly selected fields were scored.

2.4 Results.

As compared to DMSO control treatment, liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine significantlyinduced the GFP-LC3 puncta formation in HeLa cancer cells as shown inFIG. 2a . Western blot analysis showed that conversion of the autophagicmarker LC3-II was also induced upon treatments of liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezine asshown in FIG. 2b . In addition, liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine also increased the formationof GFP-LC3 puncta towards a panel of cancer and normal cells as revealedby fluorescent microscopy as shown in FIG. 2c . However, there was asignificant reduction in the liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine-autophagy induced by GFP-LC3puncta formation in HeLa cells in the presence of autophagic inhibitor(3-MA) as shown in FIG. 3, in which such findings were consistent withthe GFP-LC3 puncta formation and LC3 conversion from LC3-I to LC3-II asshown in FIGS. 2a to 2 c.

2.5 Conclusion.

The data of this study suggested that liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine are the novelautophagy enhancers. Although these compounds could induce autophagy inLO2 human normal liver cells, autophagy mediated by these compoundsexhibits far less toxic in human normal liver cells as shown in FIG. 1g, suggesting that the cytotoxic effect mediated by liensinine,isoliensinine, dauricine, cepharanthine, hernandezine or thalidezine istumor specific.

Example 3 Study on Dependency of the Presence of Autophagy-Related Gene7 (Atg7) on Autophagic Effect

This example describes an in vitro study to demonstrate that theautophagic effect of liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine is dependent on the presenceof autophagy-related gene 7 (Atg7).

3.1 Quantification of Autophagy GFP-LC3 Puncta in Atg7 Wild Type andDeficient MEFs.

GFP-LC3 puncta formation was quantified as previously described [15]. Inbrief, both Atg7 wild-type (Atg7-wt or Atg7+/+) and deficient (Atg7−/−)mouse embryonic fibroblasts (MEFs) were transfected with GFP-LC3 plasmidand then grown on coverslips in a 6-well plate. The cells were thentreated with 20 μM of liensinine, 10 μM of isoliensinine, 10 μM ofdauricine, 10 μM of cepharanthine, hernandezine or 10 μM of thalidezine.for 24 h. The cells were then fixed in 4% paraformaldehyde for 20minutes at room temperature and then rinsed with PBS. Slides weremounted with FluorSave™ mounting media (Calbiochem, San Diego, Calif.)and examined by fluorescence microscopy. The number of GFP-positivecells with GFP-LC3 puncta formation was examined under the Nikon ECLIPSE80i microscope. Representative images were captured with CCD digitalcamera Spot RT3™ (Diagnostic Instruments, Inc., Melville, N.Y.). Toquantify for autophagy, the percentage of cells with punctate GFP-LC3fluorescence was calculated by counting the number of the cells withpunctate GFP-LC3 fluorescence in GFP-positive cells. A minimum of 150cells from 3 randomly selected fields were scored.

3.2 Results:

Liensinine, isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine were found to induce GFP-LC3 puncta formation in wild typeAtg7 cells but not in Atg7-knockout (Atg7-ko or Atg7−/−) mouse embryonicfibroblasts, as shown in FIGS. 4a to 4 c.

3.3 Conclusion:

Liensinine, isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine work as the novel autophagy enhancers which depend onautophagy related gene, Atg7, for the induction of autophagy. In otherwords, the autophagy induced by the six aforementioned compounds wasAtg-7 dependent.

Example 4 Study on Mechanism of Autophagy Induction

This example describes an in vitro study to demonstrate the mechanismand action of liensinine, isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine during autophagy induction.

4.1 Detection of mTOR Signaling Marker Proteins.

HeLa cells treated with 20 μM of liensinine, 10 μM of isoliensinine, 10μM of dauricine, 10 μM of cepharanthine, 10 μM of hernandezine and 10 μMof thalidezine were harvested and lysed in RIPA buffer (Cell Signaling).The cell lysates were then resolved by SDS-PAGE. After electrophoresis,the proteins from SDS-PAGE were transferred to nitrocellulose membranewhich was then blocked with 5% non-fat dried milk for 60 minutes. Themembrane was then incubated with P-p70S6K, p70S6K, P-AMPK, AMPK andactin primary antibodies (1:1000) in TBST overnight at 4° C.respectively. After that, the membrane was further incubated withHRP-conjugated secondary antibodies for 60 minutes. Finally, proteinbands were visualized by using the ECL Western Blotting DetectionReagents (Invitrogen).

4.2 Quantification of Liensinine, Isoliensinine, Dauricine,Cepharanthine, Hernandezine and Thalidezine-Mediated Autophagy in thePresence of Specific Inhibitor.

GFP-LC3 puncta formation was quantified as previously described [15]. Inbrief, HeLa cells expressing GFP-LC3 were treated with indicatedconcentrations of liensinine, isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine in the presence of AMPK inhibitor, compoundC (CC, 5 μM), for 24 hours. The cells were then fixed in 4%paraformaldehyde for 20 minutes at room temperature and then rinsed withPBS. Slides were mounted with FluorSave™ mounting media (Calbiochem) andexamined by fluorescence microscopy. To quantify for autophagy, thepercentage of cells with punctate GFP-LC3 fluorescence was calculated bycounting the number of the cells with punctate GFP-LC3 fluorescence inGFP-positive cells. A minimum of 150 cells from 3 randomly selectedfields were scored.

4.3 Results.

Liensinine, isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine were found to activate the phosphorylation of AMPK ascompared to DMSO control treatment as shown in FIG. 5a and thisactivation was also accompanied by a concomitant reduction in itsdownstream p70S6K phosphorylation. In order to confirm whether the AMPKsignaling is involved in autophagy induced by liensinine, isoliensinine,dauricine, cepharanthine, hernandezine or thalidezine, specific AMPKinhibitor, compound C, was used in the study. Results showed that therewas a significant reduction in the GFP-LC3 puncta formation induced byliensinine, isoliensinine, dauricine, cepharanthine, hernandezine orthalidezine in HeLa cells treated with the presence of AMPK inhibitor(Compound C), as shown in FIG. 5b , suggesting that the AMPK signalingis required for autophagy induction by these alkaloid compounds.

4.4 Conclusion.

Liensinine, isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine are shown to induce autophagy via modulation of AMPK-mTORsignaling pathway.

Example 5 Study of Induction of Autophagic Cell Death in Cells

This example describes an in vitro study to demonstrate that liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezineinduce autophagic cell death in cells.

5.1 Cell Culture and Flow Cytometry Analysis.

Cell viability was measured using an annexin V staining kit (BDBiosciences, San Jose, Calif., USA). Briefly, Atg7 wild-type (Atg7 +/+or Atg7-wt) and Atg7 deficient (Atg7 −/− or Atg7-ko) mouse embryonicfibroblasts (MEFs) were treated with the selected alkaloids for 24 h.Cells were then harvested and analysed by multiparametric flow cytometryusing FITC-Annexin V and Propidium iodide staining (BD Biosciences, SanJose, Calif., USA) according to the manufacturer's instructions. Flowcytometry was then carried out using a FACSCalibur flow cytometer (BDBiosciences, San Jose, Calif., USA). Data acquisition and analysis wasperformed with CellQuest (BD Biosciences, San Jose, Calif., USA). Datawere obtained from three independent experiments.

5.2 Results.

Among the six tested compounds, as shown in FIGS. 6a to 6c , allalkaloids are found to exhibit less cytotoxic effect to autophagydeficient cells (Atg7 −/− or Atg7-ko).

5.3 Conclusion.

The results suggest that liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine induce cell death or cellcytotoxicity via autophagy induction.

Example 6 Study on Study of Induction of Cell Cytotoxicity inApoptosis-Resistant Cells

This example describes an in vitro study to demonstrate thatisoliensinine, dauricine, cepharanthine, hernandezine and thalidezinepotently induce cell cytotoxicity in apoptosis-resistant cells.

6.1 Cell Culture and Cytotoxicity Assay.

The test compounds of isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine were dissolved in DMSO at a finalconcentration of 100 mmol/L and stored at −20° C. Cytotoxicity wasassessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay as previously described [25]. 2500 of caspase wild-type(caspase WT), caspase-3 deficient (caspase 3KO), caspase-7 deficient(caspase 7KO), caspase-3/-7 deficient (caspase 3/7 DKO), caspase-8deficient (caspase 8KO), Bax-Bak wild-type (Bak-Bak WT) and Bax-Bakdouble knock out (Bak-Bak DKO) mouse embryonic fibroblasts (MEFs) wereseeded on 96-well plates per well. After overnight pre-incubation, thecells were exposed to different concentrations of isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine (0.039-100μmol/L) for 3 days. Specifically, the following concentrations are usedfor all of the above alkaloids: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625,0.78, 0.39, 0.195, 0.079, 0.039 μmol/L. Subsequently, 10 μL of MTTreagents was added to each well and incubated at 37° C. for 4 hours,followed by the addition of 100 μL solubilization buffer (10% SDS in0.01 mol/L HCl) and overnight incubation. Absorbance at 585 nm wasdetermined from each well on the following day. The percentage of cellviability was calculated using the following formula: Cell viability(%)=Cells number treated/Cells number DMSO control×100. Data wasobtained from three independent experiments.

6.2 Results.

Isoliensinine, dauricine, cepharanthine, hernandezine and thalidezineare found to exhibit similar cytotoxic effect on both wild-type andapoptosis-resistant cells, i.e. caspase-3/-7/-8 as compared to thecaspase wild-type MEFs as shown in FIGS. 7a to 7e ). In addition, fromFIGS. 7a to 7e , similar cytotoxicity is also shown in Bax-Bak DKOapoptosis-resistant cells as compared to Bax-Bak wild-type MEFs,indicating that these alkaloid compounds are able to induce cell deathin apoptosis-resistant cells.

6.3 Conclusion.

These findings suggest that isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine are capable to induce cell cytotoxicity inapoptosis-resistant cancer cells.

Example 7 Study on Clearance of Mutant Huntingtin HDQ55/74

This example describes an in vitro study to demonstrate the clearance ofmutant huntingtin HDQ55/74 by liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine.

7.1 Cell Culture and Cytotoxicity Assay.

For cell viability assay measured by crystal violet staining, PC-12cells were incubated in 35 mm disc followed by the addition ofliensinine, isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine at 5-10 μM for 24 h. The cells were then incubated withcrystal violet for 10 minutes followed by a ddH₂O wash. Images of thestained cells were captured by CCD digital camera Spot RT3™ under theNikon ECLIPSE 80 microscope with 4× magnification. Cell viability wasquantified by dissolving stained cells in 10% acetic acid (200 μL/well).The colorimetric reading of the solute mixture was then determined byspectrophotometer at OD 560 nm. The percentage of cell viability wascalculated using the following formula: Cell viability (%)=Cellsnumber_(treated)/Cells number_(DMSO control)×100. Data was obtained fromthree independent experiments.

7.2 Removal of Mutant Huntingtin.

PC 12 cells were transfected transiently with EGFP-HDQ55/74 plasmids for24 h using Lipofectamine Plus LTX reagent (Invitrogen) according to themanufacturer's protocol. The transfected cells were then treated withliensinine, isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine for 24 h. The removal of mutant huntingtin, (HDQ55& HDQ74)was then quantitated by immunoblotting with antibody against EGFP or byimmunocytochemistry under fluorescence microscopy.

7.3 Results.

As shown in FIG. 8a , liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine exhibit no toxicity in PC 12at 5-10 μM. In addition, 5-10 μM of liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine enhanced theclearance of overexpressed EGFP-tagged mutant huntingtin (HDQ55, HDQ74)with 55 and 74 CAG repeats as measured by immunoblotting against EGFPantibody as shown in FIG. 8b . Concomitantly, fluorescence imaging asillustrated in FIG. 8c further revealed that the six aforesaid compoundssignificantly reduced the formed mutant huntingtin aggregate (HDQ55) inPC 12 cells.

7.4 Conclusion.

Liensinine, isoliensinine, dauricine, cepharanthine, hernandezine andthalidezine may work as a novel neuroprotective agent throughaccelerating the clearance of mutant huntingtin.

The present invention relates to the identification of a group of novelautophagy enhancers, namely, liensinine, isoliensinine, dauricine,cepharanthine, hernandezine and thalidezine, which are isolated fromChinese medicinal herbs, Nelumbo nucifera (liensinine andisoliensinine), Asiatic Moonseed Rhizome (dauricine), Stephaniacepharantha (cepharanthine), Thalictrum hernandezii (hernandezine) andThalictrum podocarpum Humb (thalidezine) respectively. The inventionalso covers the anti-cancer effect of the above alkaloid compoundsthrough induction of autophagic cell death in a panel of cancer cellsand apoptosis-resistant cells. In addition, the invention further coversthe neuroprotective effect of the above compounds on neuronal cells viaenhancing the clearance of mutant huntingtin.

In one embodiment of the present invention, liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine exhibitsignificant cytotoxic effect towards a panel of cancer cells, but not inhuman normal liver LO2 cells. In the further embodiment of the presentinvention, liensinine, isoliensinine, dauricine, cepharanthine,hernandezine and thalidezine exhibit specific cytotoxic effect towardhuman cancer cells.

In one embodiment of the present invention, liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine are the novelautophagy enhancers and never be reported before. In the furtherembodiment of the present invention, liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine are capable toinduce autophagy in a panel of cancer and normal cells, and animals.

In one embodiment of the present invention, autophagy inducedliensinine, isoliensinine, dauricine, cepharanthine, hernandezine orthalidezine is dependent on autophagy-related gene 7 (Atg7)). In thefurther embodiment of the present invention, liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine are capable toinduce autophagy in Atg7 dependent manner.

In one embodiment of the present invention, liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine induce autophagyvia activation of AMP-activated protein kinase (AMPK) and inhibition ofmammalian target of rapamycin (mTOR) signaling. In the furtherembodiment of the present invention, liensinine, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine are capable toinduce autophagy via modulation of AMPK-mTOR signaling pathway.

In one embodiment of the present invention, liensinine, isoliensinine,dauricine and thalidezine are found to exhibit less cytotoxicity inautophagy deficient cells (Atg7−/−), indicating that liensinine,isoliensinine, dauricine and thalidezine are able to induce autophagiccell death in wild-type Atg7 cells. In the further embodiment of thepresent invention, liensinine, isoliensinine, dauricine and thalidezineare capable to induce autophagic cell death mechanism in Atg7 containingcancer cells.

In another embodiment of the present invention, isoliensinine,dauricine, cepharanthine, hernandezine and thalidezine exhibitsignificant cytotoxic effect towards a panel of apoptosis-resistantcells. In the further embodiment of the present invention,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezineexhibit potent cytotoxic effect towards apoptosis-resistant cancercells.

In another embodiment of the present invention, liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezineenhance the clearance of mutant huntingtin HDQ55/74 in PC12 cells. Inthe further embodiment of the present invention, liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezineare capable to enhance the clearance of mutant huntingtin.

The preferred embodiment of the present invention, liensinine,isoliensinine, dauricine, cepharanthine, hernandezine and thalidezinecould be developed as novel anti-cancer and neuroprotective agents forpatients with cancers or neurodegenerative diseases.

In another embodiment, the neurodegenerative diseases can be selectedfrom Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, ataxia telangiectasia, spinocerebellaratrophy and multiple sclerosis.

The exemplary embodiments of the present invention are thus fullydescribed. Although the description referred to particular embodiments,it will be clear to one skilled in the art that the present inventionmay be practiced with variation of these specific details. Hence thisinvention should not be construed as limited to the embodiments setforth herein.

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What is claimed is:
 1. A method of treating neurodegenerative disordercomprising: administering an effective amount of an alkaloid to asubject in need thereof, wherein the alkaloid is thalidezine, and theneurodegenerative disorder is Huntington's disease caused by cellscontaining Mutant huntingtin HDQ55/54.
 2. The method of claim 1, whereinthe thalidezine is represented by the following formula (I):


3. The method of claim 1, wherein the thalidezine is isolated from seedembryos of Thalictrum podocarpum Humb.